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Reproduction: intracytoplasmic sperm injection

ISSN 2398-2977


Synonym(s): ICSI

Introduction

  • Intracytoplasmic sperm injection (ICSI) is the in vitro injection of a sperm cell (spermatozoa) into the cytoplasm of a mature oocyte using a micromanipulator microscope.
  • ICSI differs from both gamete intrafallopian transfer (GIFT) Reproduction: gamete intrafallopian transfer and oocyte transfer (OT) Reproduction: oocyte transfer in that fertilization occurs outside of the female body, in a laboratory.
  • Sperm cells are sourced from fresh, chilled or frozen semen, or epididymal sperm cells collected after a stallion’s demise or castration.
  • Oocytes may be sourced from live mares Reproduction: oocyte transfer, or from deceased mares’ ovaries.
  • Oocytes can be recovered from live mares by a referring veterinarian, and shipped overnight to a laboratory for ICSI, without any decrease in oocyte or embryo viability.
  • Both germinal vesicle (GV) stage oocytes from immature follicles and mature metaphase II (MII) oocytes from pre-ovulatory follicles can be used with and without in vitro maturation (IVM) respectively.
  • ICSI in the horse was developed largely in reaction to the fact that conventional in vitro fertilization is very unsuccessful in the horse.
  • Typically, following ICSI, horse embryos are cultured in the laboratory for 7-10 days, and then those which have successfully cleaved and formed blastocysts, are then either transferred into the uterus of a synchronized recipient mare Embryo Transfer or cryopreserved for future use.
  • ICSI is a highly specialized technique which is unlikely to be performed outside of specialized Universities / referral institutions, due to the level of expertise and the equipment required. However, it is of use to practitioners to be familiar with the technique in order to be able to discuss it with their clients as a possible treatment in cases of severe stallion infertility, or of very limited sperm availability.

Uses

  • Because during ICSI fertilization takes place in vitro and the sperm cell is injected directly into the oocyte, ICSI provides a means of overcoming male fertility problems which otherwise render a stallion infertile, such as problems with sperm motility; sperm capacitation; the acrosome reaction; zona pellucida penetration and sperm-egg membrane fusion.
  • Furthermore, because only one sperm cell is need to achieve fertilization using ICSI, the technique is of use where available sperm numbers are very limited – for example if a stallion has died but some frozen sperm has been stored, or if using sperm retrieval from the epididymis of castrated or deceased horses, or if a stallion is unable to produce enough motile sperm for a standard insemination dose.
  • ICSI also overcomes certain mare infertility issues that prevent normal ovulation or oviductal transport and early embryonic maturation required for other ART procedures such as Embryo transfer. It does not however overcome mare infertility due to poor oocyte quality.

Advantages

  • Provides a method of achieving pregnancies in cases of male infertility relating to poor semen quality / limited sperm numbers and in certain mare infertility issues allowing preservation of valuable genetics which may otherwise be lost. 
  • This is useful since in other species in vitro fertilization is often used to overcome such cases of male subfertility, in horses in vitro fertilization is comparatively unsuccessful.
  • Oocytes and semen can be shipped to remote laboratories undertaking ICSI techniques, and then embryos can be transferred into a recipient Embryo transfer if normal blastocyst development results from ICSI and in vitro culture of embryos.

Disadvantages

  • Low success rates (blastocyst production following ICSI is typically <40%).

Technical problems

  • Problems with ovum pick up (OPU) and oocyte maturation
  • Need to process sperm to remove seminal plasma, debris and contamination (see below) and to select fertile sperm (see below).
  • Requires expensive equipment and very specialized expertise and knowledge of cell culture and embryological techniques.

Alternative techniques

Requirements

Personnel

Veterinarian expertise

  • Expertise in mare and stallion reproduction.
  • Expertise in recipient synchronization and embryo transfer to transfer the embryo into a recipient mare once ICSI has resulted in the successful production of a blastocyst Embryo transfer.

Other involvement

Materials required

Minimum equipment

  • See oocyte collection Reproduction: oocyte collection and oocyte transfer Reproduction: oocyte transfer for materials needed for oocyte collection, preparation and handling.
  • Sperm for ICSI are prepared by swim up, direct wash, or density-gradient or single-colloid centrifugation as a means of separating the sperm cells from the seminal plasma, debris and contamination, and of selecting morphologically and motile semen (see below).
  • Micromanipulator microscope.
  • Specialized glass capillary tubing/pipettes for handling sperm cells.
  • +/- Piezo drill.
  • Laboratory facilities to handle gametes.
  • Laboratory facilities to culture equine embryos in vitro (CO2 and mixed-gas incubators).

Minimum consumables

Other requirements

  • Stocks to restrain the mare for embryo transfer if a blastocyst develops from ICSI.

Preparation

Preparation: oocyte

Preparation: semen

  • Sperm for ICSI are prepared by swim up, direct wash, or density-gradient or single-colloid centrifugation.
  • Because the sperm cell is being deposited directly into the cytoplasm of the oocyte, seminal plasma and debris must be removed.
  • Fresh semen is washed / seminal plasma is removed by centrifugation, and resuspension in media (usually twice).
  • Where poor quality fresh or chilled semen, or frozen-thawed semen is being used, selection of motile sperm is achieved by use of a ‘swim up’ method. Briefly, a 5ml tube is filled with 1 ml of sperm-compatible media. 200µL of semen is layered into the bottom of the 5ml tube, under the media.
  • After 20 minutes incubation at 38.2°C/100.76°F, the top 600 µL of the media (into which the motile sperm will have ‘swum up’) is collected. It is centrifuged, re-suspended in media, and centrifuged again.
  • The motile sperm at this point are in a ‘sperm pellet’ at the bottom of the centrifuge tube.
  • The supernatant is removed and discarded, and the sperm pellet is gently mixed with the small amount of media that remains in the bottom of the centrifuge tube.
  • Typically, one microliter of the suspension of motile sperm that has been thus created is placed at the top of a 3µL drop of specialized sperm culture media (10% PVP) under light white mineral oil.
  • The active sperm will swim out to the edge of the PVP droplet and are subsequently selected for ICSI.

Preparation

Technique

Core procedure

  • A single sperm cell is injected into the cytoplasm of a mature (metaphase II) oocyte, under microscopic guidance.
  • This is achieved by using one pipette and gentle suction to hold the oocyte still, with the polar body located at either 12 or 6 o’clock (to avoid disrupting the metaphase plate during sperm injection).
  • A single sperm cell is immobilized either by applying one or a few Piezo pulse settings to the midpiece of the cell (if using the Piezo method), or by tail crushing (if using ‘conventional’ ICSI).
  • The sperm cell is loaded, tail first, into a second, sperm pipette (not the same as the pipette holding the oocyte in place).
  • Several sperm cells may be loaded into one pipette.
  • The sperm cell is then injected into the cytoplasm of the oocyte.
  • Where conventional ICSI is being performed, this is achieved by simply pushing the sharp-ended pipette into the cytoplasm.
  • When a Piezo drill technique is being performed, the penetration of the oocyte’s zona pellucida is achieved by drilling, with the cytoplasmic membrane being breached by Piezo pulses. Whilst there is no reported difference in blastocyst rates per injected oocyte between the two techniques (conventional pipette vs. Piezo-driven pipette), the quality of the embryos, as evidenced by nucleus number and nuclear fragmentation rate, have been shown to be significantly higher in blastocysts produced via Piezo-driven ICSI.
  • In either technique, the sperm cell is deposited deep into the oocyte cytoplasm.

Aftercare

Immediate Aftercare

  • All of the oocytes which have been injected with sperm are cultured in specialized complete cell culture media, eg Modified Dublecco’s Eagle’s medium/Ham’s F-12 (DMEM/F-12) with 10% FBS.
  • Culture is performed in 5% CO2, 5% O2, and 90% N2 at 38.2°C for 7-10 days until they form blastocysts.
  • Embryos are evaluated for cleavage on day 3 after ICSI, and blastocyst formation on days 7-10 after ICSI.
  • In vitro produced blastocysts are generally regarded to be the equivalent of early (day-6) in vivo recovered blastocysts. Therefore, in vitro produced blastocysts such should be transferred to synchronized recipient mares that are 4–6 days post ovulation on the day of transfer Embryo transfer.
  • Success of the procedure involves many complex and interdependent procedures. Successful ICSI laboratories would ideally achieve the following results; in vitro maturation rate; ≥50%, blastocyst rate per injected oocyte; ≥20%, pregnancy rate after transfer of blastocysts to recipient mares; ≥65% and foaling rate per established pregnancy; ≥80%.

Outcomes

Reasons for treatment failure

  • Poor oocyte developmental competence.
  • Failure of cleavage following ICSI.
  • Failure of blastocyst formation.
  • Early embryonic loss following ET.

Further Reading

Publications

Refereed Papers

  • Recent references from PubMed and VetMedResource.
  • Hinrichs K (2018) Assisted reproductive techniques in mares. Reprod Dom Anim 53 (2), 4–13 PubMed.
  • Choi Y H, Roasa L M et al (2004) Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Biol Reprod 70 (5), 1231-1238 PubMed.
  • Choi Y H, Love C C et al (2002) Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa. Reproduction 123 (3), 455-465 PubMed.
  • Galli C, Crotti G et al (2002) Frozen-thawed embryos produced by Ovum Pick Up of immature oocytes and ICSI are capable to establish pregnancies in the horse. Theriogenology 58 (2-4), 705-708.
  • Cochran R, Meintjes M et al (2000) Production of live foals from sperm-injected oocytes harvested from pregnant mares. J Reprod Fertil Suppl 56, 503-512 PubMed.
  • McKinnon A O, Lacham-Kaplan O & Trounson A O (2000) Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in-vivo matured oocytes. J Reprod Fertil Suppl 56, 513-517 PubMed.
  • Squires E L, Wilson J M et al (1996) A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro. Theriogenology 45, 306.

Other sources of information

  • Choi Y-H & Hinrichs K (2011) Intracytoplasmic Sperm Injection (ICSI). In: Equine Reproduction. Eds: McKinnon A O, Squires E L, Vaala W E & Varner D D. Blackwell Publishing Ltd, UK. pp 2948 – 2951
  • Hinrichs K (2010) Application of assisted reproductive technologies (ART) to clinical practice. In: Proc of the Annual Convention of the AAEP. pp 195-206.
  • McKinnon A O, Trounson A & Silber S J (2009) Intracytoplasmic Sperm Injection (ICSI). In: Proc of the 11th AAEP Annual Resort Symposium. pp 58-80.